WO2019215453A1 - Cellules car-t exprimant un anticorps à domaine unique anti-récepteur ii de tgf-bêta- inhibiteur - Google Patents

Cellules car-t exprimant un anticorps à domaine unique anti-récepteur ii de tgf-bêta- inhibiteur Download PDF

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WO2019215453A1
WO2019215453A1 PCT/GB2019/051284 GB2019051284W WO2019215453A1 WO 2019215453 A1 WO2019215453 A1 WO 2019215453A1 GB 2019051284 W GB2019051284 W GB 2019051284W WO 2019215453 A1 WO2019215453 A1 WO 2019215453A1
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seq
cell
antibody
cells
receptor
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PCT/GB2019/051284
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Martin Pule
Shaun CORDOBA
Simon Thomas
Shimobi ONUOHA
Alex KINNA
Mathieu FERRARI
Marco DELLA PERUTA
Phillip Wu
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Autolus Limited
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Priority to US17/053,597 priority Critical patent/US20210244762A1/en
Priority to EP19724579.8A priority patent/EP3790583A1/fr
Publication of WO2019215453A1 publication Critical patent/WO2019215453A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464469—Tumor associated carbohydrates
    • A61K39/464471—Gangliosides, e.g. GM2, GD2 or GD3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to a cell which comprises a chimeric antigen receptor (CAR) or transgenic T cell receptor (TOR) and secretes a factor which binds a transforming growth factor beta receptor O ⁇ R).
  • the factor may be an antibody.
  • Adoptive immunotherapy of cancer involves the ex vivo generation of cancer-antigen specific cells and their administration.
  • Adoptively transferred immune effector cells also activate existing adaptive and innate immune cells within the tumour once they activate and start causing inflammation.
  • the native specificity of immune effector cells can be exploited in adoptive immunotherapy - for example during the generation of melanoma specific T-cells from expansion of tumour infiltrating lymphocytes in tumour resections. Otherwise a specificity can be grafted onto a T- cell using genetic engineering. Two common methods for achieving this are using chimeric antigen receptors or transgenic T-cell receptors. Different kinds of immune effector cells can also be used. For example, alpha/beta T-cells, NK cells, gamma delta T-cells or macrophages can be used.
  • B-ALL B-cell Acute Lymphoblastic Leukaemia
  • DLBCL Diffuse Large B-cell Lymphoma
  • MM Multiple Myeloma
  • tumour microenvironment may convert the microenvironment into a more favourable environment which enables the engineered immune effector cells to proliferate, survive and/or engraft thereby providing a more effective engineered cell therapy.
  • TQRb transforming growth factor beta
  • Fresolimumab is a neutralizing antibody which blocks TQRb1-3. Fresolimumab has been tested in metastatic melanoma and high-grade glioma. This showed some effectiveness in the enhancement of a tumour-specific immune response but failed to eradicate the tumour.
  • Other approaches include small molecules which inhibit SMAD signalling, downstream of transforming growth factor beta receptor (TbR).
  • TbR transforming growth factor beta receptor
  • Galunisertib which has been tested as a monotherapy or in combination with alkylating agents, Lomustine or temozolamide for glioblastoma and other combinations. These approaches have focussed on the inhibitory microenvironment and have not been particularly effective.
  • the present inventors have developed a cell with an in-built system to control TQRb signalling, rendering the cell less susceptible (i.e. more resistant) to TQRb.
  • the present invention provides a cell which comprises a chimeric antigen receptor (CAR) or transgenic T cell receptor (TOR) and secretes a factor which binds a transforming growth factor beta receptor (TbR).
  • CAR chimeric antigen receptor
  • TOR transgenic T cell receptor
  • TbR transforming growth factor beta receptor
  • the secreted factor may block the interaction between TbR and TQRb.
  • the secreted factor may bind TGF-beta receptor type-2 (TbRII).
  • the secreted factor may comprise a variant T ⁇ Rb polypeptide, a mutant T ⁇ Rb or a truncated T ⁇ Rb polypeptide.
  • the secreted factor may be or comprise an antibody.
  • the antibody may be a domain antibody (dAb), for example a dAb which comprises one of the following sets of complementarity determining regions (CDRs):
  • dAb domain antibody
  • CDRs complementarity determining regions
  • the cell may secrete a dAb comprising one of the sequences shown as SEQ ID No. 13, 14, 15 or 16.
  • the present invention provides a domain antibody (dAb) which binds transforming growth factor beta receptor type II (TbRII) and comprises one of the following sets of complementarity determining regions (CDRs):
  • dAb domain antibody
  • TbRII transforming growth factor beta receptor type II
  • CDRs complementarity determining regions
  • the dAb may comprise one of the sequences shown as SEQ ID No. 13, 14, 15 or 16.
  • the present invention provides a nucleic acid sequence encoding a dAb according to the second aspect of the invention.
  • the present invention provides a nucleic acid construct which comprises: (i) a first polynucleotide which encodes a secreted factor or antibody as defined in the first aspect of the invention; and (ii) a second polynucleotide which encodes a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TCR).
  • a nucleic acid construct which comprises: (i) a first polynucleotide which encodes a secreted factor or antibody as defined in the first aspect of the invention; and (ii) a second polynucleotide which encodes a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TCR).
  • CAR chimeric antigen receptor
  • TCR transgenic T-cell receptor
  • the first and second polynucleotides may be separated by a co-expression site.
  • the present invention provides a kit of polynucleotides comprising:
  • a second polynucleotide which encodes a chimeric antigen receptor (CAR) or a transgenic (TCR).
  • the present invention provides a vector which comprises a nucleic acid sequence according to the third aspect of the invention or a nucleic acid construct according to the fourth aspect of the invention.
  • kit of vectors which comprises:
  • a first vector which comprises a polynucleotide which encodes a secreted factor or antibody as defined in the first aspect of the invention.
  • a second vector which comprises a polynucleotide which encodes a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TCR).
  • CAR chimeric antigen receptor
  • TCR transgenic T-cell receptor
  • the present invention provides a pharmaceutical composition which comprises a cell according to the first aspect of the invention or a dAb according to the second aspect of the invention.
  • the present invention provides a pharmaceutical composition according to the eighth aspect of the invention, for use in treating and/or preventing a disease.
  • the present invention provides a method for treating and/or preventing a disease, which comprises the step of administering a pharmaceutical composition according to the eighth aspect of the invention to a subject in need thereof.
  • the present invention provides the use of a cell according to the first aspect of the invention, or a dAb according to the second aspect of the invention in the manufacture of a medicament for the treatment and/or prevention of a disease.
  • the disease may be cancer.
  • the present invention provides method for making a cell according to the first aspect of the invention, which comprises the step of introducing: a nucleic acid sequence according to third aspect of the invention, or a nucleic acid construct according to the fourth aspect of the invention, a first polynucleotide and a second polynucleotide as defined in the fifth aspect of the invention, a vector according to the sixth aspect of the invention or a first and second vector as defined in the seventh aspect of the invention into a cell ex vivo.
  • a cell which comprises a chimeric antigen receptor (CAR) or transgenic T cell receptor (TCR) and secretes a domain antibody (dAb).
  • CAR chimeric antigen receptor
  • TCR transgenic T cell receptor
  • dAb domain antibody
  • a cell according to paragraph A1 wherein the dAb binds transforming growth factor beta (TQRb) or a TQRb receptor 0W, PD-1 , PD-L1 , LAG-3 or CTLA-4.
  • TQRb transforming growth factor beta
  • a nucleic acid construct which comprises: (i) a first polynucleotide which encodes a domain antibody (dAb); and (ii) a second polynucleotide which encodes a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TCR).
  • dAb domain antibody
  • CAR chimeric antigen receptor
  • TCR transgenic T-cell receptor
  • a kit of polynucleotides comprising:
  • a second polynucleotide which encodes a chimeric antigen receptor (CAR) or a transgenic (TCR).
  • a kit of vectors which comprises:
  • a first vector which comprises a polynucleotide which encodes a domain antibody (dAb);
  • a second vector which comprises a polynucleotide which encodes a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TCR).
  • CAR chimeric antigen receptor
  • TCR transgenic T-cell receptor
  • a pharmaceutical composition which comprises a cell according to paragraph A1 or A2.
  • a pharmaceutical composition according to paragraph A7 for use in treating and/or preventing a disease.
  • a method for treating and/or preventing a disease which comprises the step of administering a pharmaceutical composition according to paragraph A7 to a subject in need thereof.
  • a method for making a cell according to paragraph A1 oe A2 which comprises the step of introducing: a nucleic acid construct according to paragraph A3, a first polynucleotide and a second polynucleotide as defined in paragraph A4, a vector according to paragraph A5 or a first and second vector as defined in paragraph A6 into a cell ex vivo.
  • a cell which comprises a chimeric antigen receptor (CAR) or transgenic T cell receptor (TCR) and secretes a Fab antibody (Fab).
  • CAR chimeric antigen receptor
  • TCR transgenic T cell receptor
  • Fab Fab
  • a cell according to paragraph B1 wherein the Fab antibody binds transforming growth factor beta (TQRb) or a TQRb receptor (TbR), PD-1 , PD-L1 , LAG-3 or CTLA-4.
  • TQRb transforming growth factor beta
  • TbR TQRb receptor
  • a nucleic acid construct which comprises: (i) a first polynucleotide which encodes a Fab antibody (Fab); and (ii) a second polynucleotide which encodes a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TCR).
  • Fab Fab antibody
  • CAR chimeric antigen receptor
  • TCR transgenic T-cell receptor
  • a kit of polynucleotides comprising:
  • a second polynucleotide which encodes a chimeric antigen receptor (CAR) or a transgenic (TCR).
  • a kit of vectors which comprises:
  • a first vector which comprises a polynucleotide which encodes a Fab antibody (Fab);
  • a second vector which comprises a polynucleotide which encodes a chimeric antigen receptor (CAR) or a transgenic T-cell receptor (TCR).
  • CAR chimeric antigen receptor
  • TCR transgenic T-cell receptor
  • a pharmaceutical composition according to paragraph B7, for use in treating and/or preventing a disease is provided.
  • a method for treating and/or preventing a disease which comprises the step of administering a pharmaceutical composition according to paragraph B7 to a subject in need thereof.
  • a method for making a cell according to paragraph B1 or B2 which comprises the step of introducing: a nucleic acid construct according to paragraph B3, a first polynucleotide and a second polynucleotide as defined in paragraph B4, a vector according to paragraph B5 or a first and second vector as defined in paragraph B6 into a cell ex vivo.
  • the present invention provides cells which secrete a secreted factor or antibody which binds a transforming growth factor beta receptor (TbB).
  • TbB transforming growth factor beta receptor
  • the cells of the present invention are capable of modulating the inhibitory microenvironment and preventing inhibition of immune effector cells, thereby augmenting the ability of CAR- or TCR- expressing cells to attack the tumour.
  • the cells of the present invention will also support other cells which are known to be affected by TQRb, including B-cells (IgA class switching and promotes decrease in activation and apoptosis), NK cells (decreased cytotoxicity and decreased chemotaxis), neutrophils (decreased effector function and promotes N1 to N2 differentiation), macrophages (decrease effector function, decreased antigen presentation and increased inflammatory cytokine secretion as well as promoting M2 differentiation over M1) and finally dendritic cells (decreased maturation, decreased antigen presentation and decreased chemotaxis).
  • B-cells IgA class switching and promotes decrease in activation and apoptosis
  • NK cells decreased cytotoxicity and decreased chemotaxis
  • neutrophils decreased effector function and promotes N1 to N2 differentiation
  • macrophages decrease effector function, decreased antigen presentation and increased inflammatory cytokine secretion as well as promoting M2 differentiation over M1
  • FIG. 1 Schematic showing different generations of chimeric antigen receptors. The basic architecture of a canonical CAR is shown as well as different iterations of the three generations of this form of receptor.
  • FIG. 2 Schematic showing a CAR T-cell with enhanced with TQRb resistance according to the present invention.
  • a CAR T-cell secretes a factor which binds to part of the receptor complex of TQRb.
  • inhibitory effects on the CAR T-cell itself are blocked. Further, inhibitory effects on surrounding cells are also blocked.
  • FIG. 3 Schematic showing the mechanism of TQRb signalling.
  • Panel (a) shows a receptive cell expressing both type I and II receptors which are in solution in the cell membrane. They are not associated.
  • Panel (b) shows a secreted activated TQRb which forms a homodimer which binds two type I and two type II receptors. This complex results in the approximation of the two receptors which then results in their phosphorylation and down stream signalling
  • FIG. 4 Schematic showing an embodiment of the present invention comprising dominant negative TQRb.
  • Panel (a) shows normal activation by TQRb.
  • Panel (b) shows a dominant negative TQRb which has been generated by mutating binding sites for one of the receptor types (shown with black arrows). Binding does not result in signalling since association between type I and II receptors does not occur. Binding of wild-type TQRb is prevented.
  • FIG. 5 Schematic showing an embodiment of the present invention comprising a truncated dominant negative TQRb.
  • Panel (a) shows normal activation by TGF.
  • Panel (b) shows a variant T ⁇ Rb which comprises mutations and deletions that prevent homodimerization and prevent signalling.
  • Figure 6 Schematic showing an embodiment of the present invention comprising blocking of T ⁇ Rb by scFv (panel (b)). Panel (a) shows normal activation by TGF.
  • Figure 7 Schematic showing an embodiment of the present invention comprising blocking of TQRb by a dAb (panel (b)). Panel (a) shows normal activation by TGF.
  • A. A schematic diagram illustrating the incremental response units (RU) of the sensograms.
  • First RU increase red represents anti-TGFbRII VHH-Fc binding to the chip surface.
  • Second RU increase green represents soluble TGFbRII binding to the captured antibody.
  • the third RU increase blue represents the binding of TGFbeta 1 to the captured receptor.
  • FIG 11 Flow cytometry assay showing binding of the anti-TGFbRII dAbs (C6, G5, E11 and H3) to cells expressing dominant negative TGFbRII.
  • Figure 12 Cytotoxicity assay showing the % survival of target cells following 5 days co culture with CAR-expressing cells which either secrete no antibody or secrete one of the anti-TGFbRII dAbs (C6, G5, E11 or H3) in the presence or absence of soluble T ⁇ Rb1.
  • TGF-b Transforming growth factor beta
  • the transforming growth factor beta receptors are a superfamily of serine/threonine kinase receptors. These receptors bind members of the T ⁇ Rb superfamily of growth factor and cytokine signalling proteins. There are five type II receptors (which are activatory receptors) and seven type I receptors (which are signalling propagating receptors). Type I receptors are also known as activin receptor-like kinases (ALKS).
  • TGF-beta receptor type-1 (TbRI) is available from UniProt accession P36897 is shown below as SEQ ID NO: 17.
  • TGF-beta receptor type-2 (TbRII) is available from UniProt accession P37173 is shown below as SEQ ID NO: 18.
  • Variant sequences of SEQ ID NO: 17 and 18 may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 17 and 18 respectively.
  • the variant sequence of SEQ ID NO: 17 or 18 is able to function as T ⁇ Rb receptor.
  • T ⁇ Rb1 and 2 are implicated in cancer, where they may stimulate the cancer stem cell, increase fibrosis / desmoplastic reactions and suppress immune recognition of the tumour.
  • TQRb1 , 2 and 3 signal via binding to receptors T RII and then association to TbRI and in the case of TQRb2 also to TbRIII. This leads to subsequent signalling through SMADs via TbRI.
  • TQRbe are typically secreted in the pre-pro-form.
  • The“pre” is the N-terminal signal peptide which is cleaved off upon entry into the endoplasmic reticulum (ER).
  • The“pro” is cleaved in the ER but remains covalently linked and forms a cage around the TQRb called the Latency Associated Peptide (LAP).
  • LAP Latency Associated Peptide
  • the cage opens in response to various proteases including thrombin and metalloproteases amongst others.
  • the C-terminal region becomes the mature TQRb molecule following its release from the pro-region by proteolytic cleavage.
  • the mature TQRb protein dimerizes to produce an active homodimer.
  • the TQRb homodimer interacts with a LAP derived form the N-terminal region of the TQRb gene product, forming a complex called Small Latent Complex (SLC).
  • SLC Small Latent Complex
  • This complex remains in the cell until it is bound by another protein, an extracellular matrix (ECM) protein called Latent TQRb binding protein (LTBP) which together forms a complex called the large latent complex (LLC). LLC is secreted to the ECM.
  • ECM extracellular matrix
  • LTBP Latent TQRb binding protein
  • LLC large latent complex
  • LLC is secreted to the ECM.
  • TQRb is released from this complex to a biologically active form by several classes of proteases including metalloproteases and thrombin.
  • TQRb cancerous tumour cells
  • TQRb can be produced by the wide variety of non- cancerous cells present at the tumour site. Specifically, tumour-associated T cells, natural killer (NK) cells, macrophages, epithelial cells and stromal cells have all been shown to produce TGF-b in various tumour models.
  • NK natural killer
  • the present invention provides a cell comprising a polynucleotide encoding a secreted factor which is capable of binding a transforming growth factor beta receptor O ⁇ R) and disrupting its interaction with transforming growth factor beta (TQRb).
  • secreted factor is a protein which is secreted by a cell.
  • protein and polypeptide are used synonymously herein.
  • Secretory proteins typically comprise an N-terminal signal peptide so that when the secretory protein is expressed inside a cell, the nascent protein is directed to the ER.
  • the classical protein secretion pathway is through the endoplasmic reticulum (ER). VARIANT TQRb
  • the secreted factor comprises a dominant negative TQRb.
  • Dominant negative TQRb or dnTGFb as used herein means that the secreted factor TQRb acts antagonistically to the wild-type TQRb.
  • variants of TQRb as disclosed herein may inhibit the function of their natural or wild-type counterparts.
  • the variants of TQRb may inhibit signalling induced by wild- type TQRb and thus neutralise its biological effects.
  • binding of the dnTGFb to Tb does not induce productive signalling downstream of Tb .
  • the secreted factor comprises a variant T ⁇ Rb polypeptide.
  • a variant T ⁇ Rb encompasses a mutant T ⁇ Rb or truncated T ⁇ Rb as described herein.
  • variant T ⁇ Rb polypeptide means the polypeptide has an amino acid sequence which has one, two, three or more additions, deletions and/or substitutions compared with the wild-type T ⁇ Rb polypeptide.
  • the variant T ⁇ Rb polypeptide has less than 100% sequence identity to a wild-type T ⁇ Rb polypeptide.
  • the variant T ⁇ Rb polypeptide may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 99% sequence identity to a wild-type T ⁇ Rb polypeptide.
  • the variant T ⁇ Rb is able to bind a Tb and disrupt its interaction with TGF$.
  • the percentage identity between two polypeptide sequences may be readily determined by programs such as BLAST, which is freely available at http://blast.ncbi.nlm.nih.gov. Suitably, the percentage identity is determined across the entirety of the reference and/or the query sequence.
  • the wild-type polypeptide of T ⁇ Rb may comprise a signal peptide and a latency associated pro-peptide.
  • the wild-type polypeptide of T ⁇ Rb may comprise a signal peptide.
  • the secreted factor may comprise a variant mature T ⁇ Rb polypeptide.
  • the wild-type T ⁇ Rb polypeptide may be T ⁇ Rb1.
  • the wild-type T ⁇ Rb polypeptide may comprise the amino as set forth in UniProt accession P01137.
  • the amino acid sequence of wild-type TQRb1 from UniProt accession P01137 is set forth in SEQ ID NO: 19.
  • the wild-type TQRb may comprise the sequence set forth in SEQ ID NO: 1. SEQ ID NO: 19.
  • VGRKPKVEQLSNMIVRSCKCS SEQ ID NO: 19
  • the secreted factor may comprise a variant of SEQ ID NO: 19.
  • the wild-type T ⁇ Rb polypeptide may be T ⁇ Rb2.
  • the wild-type T ⁇ Rb polypeptide may comprise the amino as set forth in UniProt accession P61812.
  • the amino acid sequence of wild-type T ⁇ Rb2 from UniProt accession P61812 is set forth in SEQ ID NO: 20.
  • the wild-type T ⁇ Rb may comprise the sequence set forth in SEQ ID NO: 20.
  • SEQ ID NO: 20 comprises a pre-pro-sequence.
  • the secreted factor may comprise a variant of SEQ ID NO: 20.
  • the signal peptide of wild-type T ⁇ Rb2 which is comprise in SEQ ID NO: 20 is set forth in SEQ ID NO: 21.
  • MHYCVLSAFLILHLVTVALS (SEQ ID NO: 21)
  • the signal peptide may comprise the amino acid sequence set forth in SEQ ID NO: 21 or a variant thereof.
  • the variant signal peptide may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 21 , provided that the sequence provides an effective signal peptide. That is, provided that the polypeptide is capable of directing a newly synthesized secreted factor to the secretory pathway.
  • the latency-associated peptide fragment of wild-type TQEb2 is set forth in SEQ ID NO: 22.
  • the latency-associated peptide may comprise the amino acid sequence set forth in SEQ ID NO: 22 or a variant thereof.
  • the variant latency associated peptide may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 22, provided that the peptide is capable of interacting with T ⁇ Rb homodimer and forming a small latent complex.
  • the mature protein of wild-type T ⁇ Rb2 is set forth in SEQ ID NO: 23.
  • the secreted factor may comprise a variant of SEQ ID NO: 23.
  • the wild-type T ⁇ Rb polypeptide may be T ⁇ Rb3.
  • the wild-type T ⁇ Rb polypeptide may comprise the amino as set forth in UniProt accession P10600.
  • the amino acid sequence of wild-type T ⁇ Rb3 from UniProt accession P10600 is set forth in SEQ ID NO: 24.
  • the wild-type T ⁇ Rb may comprise the sequence set forth in SEQ ID NO: 24.
  • the secreted factor may comprise a variant of SEQ ID NO: 24.
  • the secreted factor may comprise a variant of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23 or SEQ ID NO: 24, wherein the variant has less than 100% sequence identity to SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23 or SEQ ID NO: 24.
  • the variant may have at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 99% sequence identity to SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 23 or SEQ ID NO: 24.
  • the secreted factor comprises a mutant T ⁇ Rb.
  • the mutant T ⁇ Rb is capable of binding a transforming growth factor beta receptor (TbR) and disrupting its interaction with transforming growth factor beta (T ⁇ Rb).
  • TbR transforming growth factor beta receptor
  • a“mutant T ⁇ Rb” is a T ⁇ Rb protein which comprises one or more amino acid mutations with respect to the wild-type T ⁇ Rb polypeptide.
  • a mutant T ⁇ Rb may bind TbR1 or TbRII and prevent the approximation of both receptors, thereby inhibiting signalling downstream of TbR.
  • a mutant T ⁇ Rb may comprise a mutation which eliminates or decreases their ability to signal through the ALK5 (TbRI) receptor.
  • Such mutants maintain their ability to bind with high affinity to TbRII and TbRIII receptors, but are unable to signal because they do not interact with the ALK5 (TbRI) receptor, negatively modulating wild-type T ⁇ Rb signalling by competing with them for binding to high affinity TbRII and TbRIII receptors.
  • the mutated T ⁇ Rb maintains a binding affinity to TbRII of at least the binding affinity of wild-type T ⁇ Rb1 and T ⁇ Rb3.
  • the mutant TQRb comprises mutations which increase their affinity for the receptor T RI I and/or T RIII.
  • the mutation may be at the interaction interface with the receptor.
  • the mutant TQRb may comprise one or more mutations with respect to SEQ ID NO: 19.
  • the mutant TQRb may comprise one or more mutations with respect to SEQ ID NO: 20.
  • the mutant TQRb may comprise one or more mutations with respect to SEQ ID NO: 23.
  • the mutant TQRb may comprise one or more mutations with respect to SEQ ID NO: 24.
  • the mutant TQRb may comprise one, two, three, four five or six mutations.
  • the mutations are selected to have different physicochemical properties with respect to the amino acid present in the wild-type polypeptide.
  • the mutation may be a change in an amino acid residue from nonpolar to polar, from charged to uncharged from large to small or from acid to basic.
  • the mutant TQRb may comprise one or more mutations at positions selected from amino acid residues: 30, 43, 101 , 51 , 67 or 6; when the amino acid number is determined by alignment with SEQ ID NO: 23.
  • the amino acid numbering corresponds with the numbering of amino acids as set forth in SEQ ID NO: 23.
  • the mutant TQRb may comprise one or more mutations at positions selected from amino acid residues: W30, W32, L101 , L51 , Q67 and Y6 when the amino acid number is determined by alignment with SEQ ID NO: 23. These amino acid residues of TQRb have been identified as important for TbRI interaction and important in stable binding.
  • the mutant TQRb may comprise one or more mutations, for example two, three, four, five or six mutations, at amino acid residues W30, W32, L101 , L51 , Q67 and Y6 when the amino acid number is determined by alignment with SEQ ID NO: 23, wherein:
  • amino acid residue 30 is mutated to N,R,K,D,Q,L,S,P,V,I,G,C,T,A or E; and/or
  • amino acid residue 32 is mutated to A;
  • amino acid residue 101 is mutated to A, E; and/or
  • amino acid residue 51 is mutated to Q,W,Y,A;
  • amino acid residue 67 is mutated to H, F, Y, W, Y;
  • mutant TQEb may comprise mutations at each of amino acid residues W30, W32, L101 , L51 , Q67 and Y6 when the amino acid number is determined by alignment with SEQ ID NO: 23.
  • amino acid substitutions may be selected from the list above.
  • the mutant TQEb may comprise the following mutations: W30E, L101 E and L51Q when the amino acid number is determined by alignment with SEQ ID NO: 23.
  • the mutant TQRb may comprise the following mutations: W30E, L101A and L51Q when the amino acid number is determined by alignment with SEQ ID NO: 23.
  • the mutant TQRb may comprise the following mutations: W30E, L101 E, L51Q and Q67H when the amino acid number is determined by alignment with SEQ ID NO: 23.
  • the mutant TQRb may comprise the following mutations: W30E, L101A, K97D, E12H, L51Q and Q67H when the amino acid number is determined by alignment with SEQ ID NO: 23.
  • the secreted factor comprises a truncated TQRb polypeptide.
  • the truncation may comprise an N-terminal deletion with respect to a wild-type TQRb polypeptide.
  • the truncation may comprise a C-terminal deletion with respect to a wild-type polypeptide.
  • the truncation may comprise a deletion of 1 , 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acids.
  • the secreted factor is monomeric TQRb.
  • the secreted factor may be unable to homodimerize, thereby inhibiting signalling downstream of TbE.
  • the secreted factor may prevent association between TbEI and TbEII receptors.
  • the secreted factor may be a TQRb which lacks the heel helix a3, a structural motif essential for binding the TQRb type I receptor (TbEI) but dispensable for binding TbEII.
  • the secreted factor may be a TQEb which lacks the heel helix a3, a structural motif essential for binding the TQEb type I receptor (TbEI) but dispensable for binding TbEII and which lacks Cys-77.
  • the amino acid sequence of a TQRb monomer is set forth in SEQ ID NO: 25. SEQ ID NO:
  • LAP 25 comprises a signal peptide and a latency associated peptide (LAP).
  • VGRKPKVEQLSNMIVKSCKCS SEQ ID NO: 25.
  • the secreted factor may comprise an amino acid sequence set forth in SEQ ID NO:
  • the variant T ⁇ Rb monomer may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 25, provided that the polypeptide provides a monomer which is capable of binding a transforming growth factor beta receptor (TbR) and disrupting its interaction with transforming growth factor beta (T ⁇ Rb).
  • TbR transforming growth factor beta receptor
  • T ⁇ Rb transforming growth factor beta
  • the secreted factor may comprise or consist of the amino acid sequence set forth in SEQ ID NO: 25.
  • amino acid sequence of the signal peptide comprised within SEQ ID NO: 25 is set forth in SEQ ID NO: 26.
  • the signal peptide may comprise the amino acid sequence set forth in SEQ ID NO:
  • the variant signal peptide may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 26, provided that the sequence provides an effective signal peptide. That is, provided that the sequence is capable of directing a newly synthesized secreted factor to the secretory pathway.
  • the secreted factor may be an antibody or a variant thereof.
  • the antibody may be a full-length antibody, a single chain antibody fragment, a F(ab) fragment, a F(ab’) 2 fragment, a F(ab’) fragment, a single domain antibody (sdAb), a VHH/nanobody, a nanobody, an affibody, a fibronectin artificial antibody scaffold, an anticalin, an affilin, a DARPin, a VNAR, an iBody, an affimer, a fynomer, a domain antibody (DAb), an abdurin/ nanoantibody, a centyrin, an alphabody or a nanofitin which is capable of binding a TbR and disrupting its interaction with TQRb.
  • sdAb single domain antibody
  • VHH/nanobody a nanobody
  • an affibody a fibronectin artificial antibody scaffold
  • an anticalin an affilin, a DARPin
  • VNAR an iBody
  • TQRb antibodies are well known in the art.
  • a variant secreted factor according to the present invention may have, for example, one, two or three or more amino acid mutations, for example one, two or three or more amino acid substitutions with respect to the amino acid sequences of secreted factors disclosed herein, for example in SEQ ID NO: 25 or SEQ ID NO: 26.
  • the amino acid substitutions are conservative substitutions.
  • amino acids with uncharged polar head groups having similar hydrophilicity values include leucine, isoleucine, valine, glycine, alanine, asparagine, glutamine, serine, threonine, phenylalanine, and tyrosine.
  • the present invention also encompasses homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue, with an alternative residue) i.e. like-for-like substitution such as basic for basic, acidic for acidic, polar for polar etc. Unless otherwise explicitly stated herein by way of reference to a specific, individual amino acid, amino acids may be substituted using conservative substitutions as recited below.
  • An aliphatic, non-polar amino acid may be a glycine, alanine, proline, isoleucine, leucine or valine residue.
  • An aliphatic, polar uncharged amino may be a cysteine, serine, threonine, methionine, asparagine or glutamine residue.
  • An aliphatic, polar charged amino acid may be an aspartic acid, glutamic acid, lysine or arginine residue.
  • An aromatic amino acid may be a histidine, phenylalanine, tryptophan or tyrosine residue.
  • Variants may maintain their ability to bind to TbRII.
  • the ability of a variant TQRb to bind to a TbRII may be measured by any means known in the art for example by an ELISA assay, to detect TbEII receptor chain.
  • Variant TQRb may block the binding of wild-type TQRb ligands to TbEII. This may be measured by a competitive ELISA covering the plates with TQRb ligand and assessing the ability of the variant to inhibit TbEII binding to the ligands.
  • Variant TQRb may have reduced ability to signal through TbEI or may not be capable of signalling through TbEI. This may be measured by western blotting and quantifying the levels and ratios of phosphorylated SMAD2 and SMAD3. Mutant TQRb may induce 100 times less phosphorylation than wild-type TQRb.
  • Variant TQRb may be are capable of inhibiting signalling induced by wild-type TQRb. This may be measured by Western immunoblotting assays and quantifying the levels of phosphorylated SMAD2 and SMAD3 in cell lysates treated with the mutant or wild-type TQRb.
  • the present invention provides cell which comprises a chimeric antigen receptor (CAR) or transgenic T cell receptor (TCR) and secretes an antibody which binds a transforming growth factor beta receptor 0 ⁇ R).
  • the antibody may be capable of binding a transforming growth factor beta receptor 0 ⁇ R) and disrupting its interaction with transforming growth factor beta (TQRb).
  • the antibody may be a full-length antibody or a fragment thereof such as a single chain antibody fragment, a F(ab) fragment, a F(ab’)2 fragment, a F(ab’) fragment, a domain antibody (dAb), a VHH/nanobody, a nanobody, a VNAR or IgNAR.
  • antibody includes antibody mimetics such as an affibody, a fibronectin artificial antibody scaffold, an anticalin, an affilin, a DARPin, an iBody, an affimer, a fynomer, an abdurin/ nanoantibody, a centyrin, an alphabody a nanofitin or a D-domain which are capable of binding a transforming growth factor beta receptor (TbR) and disrupting its interaction with transforming growth factor beta (TQRb).
  • TbR transforming growth factor beta receptor
  • TQRb transforming growth factor beta
  • the antibody may be a dAb comprising one of the following sets of complementarity determining regions (CDRs):
  • the dAb may comprise one of the sequences shown as SEQ ID No. 13, 14, 15 or 16.
  • the antibody may comprise a variant of SEQ ID No. 13 to 16. Variant sequences may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 13, 14, 15 or 16.
  • the variant sequence may comprise one of the sets of CDRs labelled a), b), c) or d) above.
  • the variant antibody should retain the capacity to bind a transforming growth factor beta receptor (TbR)
  • TbR ⁇ secreted factor or antibody by the cell of the invention may be “tunable”.
  • tunable means that it is possible to increase, decrease, turn on or turn off secretion or activity of the secreted factor or antibody.
  • Production of the a secreted factor or antibody may be controlled or tuned by an inducible promoter.
  • production of the a secreted factor or antibody may be regulated by Nuclear factor of activated T cells (NFAT) response element.
  • NFAT Nuclear factor of activated T cells
  • An NFAT response element may comprise the nucleotide sequence set forth in SEQ ID NO: 27 or a variant thereof.
  • Variant sequences of SEQ ID NO: 27 may have at least 80%, 85%, 90%, 95%, 98% or 99% sequence identity to SEQ ID NO: 27.
  • the variant sequence is able to function as a NFAT response element.
  • the NFAT response element may comprise repeat units such as 3, 4, 5 or 6 repeat units.
  • the NFAT response element may comprise 3, 4, 5 or 6 repeat units of SEQ ID NO: 27.
  • the NFAT response element may be positioned in front of a promoter (e.g. a CMV promoter).
  • Secretion of the secreted factor or antibody may be controlled or tuned through interaction with an intracellular retention domain.
  • An agent may be used to disrupt the interaction with the intracellular retention domain, thereby allowing secretion of the secreted factor or antibody from the cell.
  • the activity of the secreted factor or antibody may be controlled or tuned.
  • Activity as used herein means the ability of the secreted factor or antibody to bind a transforming growth factor beta receptor (TbB) thereby disrupting its interaction with transforming growth factor beta (TQRb).
  • TbB transforming growth factor beta receptor
  • TQRb transforming growth factor beta
  • the activity of the secreted factor or antibody may be inhibited by providing a sink which binds the factor, preventing or reducing interaction between the secreted factor or antibody and the receptor.
  • a sink which binds the factor
  • blockade of the secreted factor or antibody with antibodies or soluble Tb ⁇ may be used to control or tune the activity of the antibody.
  • “disrupted” or“disruption” means that the binding between Tb ⁇ and TQRb is reduced or eliminated completely by the secreted factor or antibody.
  • binding between Tb ⁇ and TQRb may be reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% by the secreted factor or antibody.
  • binding between Tb ⁇ and TQRb may be eliminated.
  • Tb ⁇ and TQRb may be disrupted by the secreted factor or antibody binding competitively to TbB.
  • competitive binding of the secreted factor or antibody refers to the binding of a secreted factor or antibody which prevents binding between Tb ⁇ and TQRb.
  • the secreted factor or antibody may bind competitively by directly binding to binding site of Tb ⁇ which interacts with the reciprocal binding site on TQRb.
  • the secreted factor or antibody may bind competitively by binding to a region which overlaps with the binding site of TbR which interacts with the reciprocal binding site on TQRb.
  • the secreted factor or antibody may be capable of specifically binding TbR at a higher affinity than the binding between the TbR and TQRb.
  • “higher affinity” means that the secreted factor or antibody binds to TbR with at least 5, 10, 20, 50, 100, 1000 or 10000-fold greater affinity than the binding affinity between TbR and TQRb.
  • radioactive ligand binding assays including saturation binding, scatchard plot
  • non radioactive ligand binding assays including fluorescence polarization, fluorescence resonance energy transfer and surface plasmon resonance/Biacore
  • solid phase ligand binding assays Any method known in the art may be used to measure binding affinity of the antibody.
  • Binding of the secreted factor or antibody may prevent the approximation of TbBI and TbBII.
  • Binding of the secreted factor or antibody may reduce or eliminate signalling downstream of TbB.
  • signalling downstream of TbB may be reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99% by the secreted factor.
  • signalling downstream of TbB may be eliminated.
  • Assays for measuring downstream signalling of TbB are known in the art such as luminescent kinase assays which measure ADP formed from the kinase reaction or measuring the proportion of cytoplasmic signalling molecules such as SMAD/SMAD2 phosphorylation. Any method known in the art may be used to measure downstream signalling of TbR.
  • the present invention relates to cell which comprises a chimeric antigen receptor (CAR) or transgenic T cell receptor (TCR) and secretes an secreted factor or antibody which binds a transforming growth factor beta receptor 0 ⁇ R)
  • CAR chimeric antigen receptor
  • TCR transgenic T cell receptor
  • the cell is "engineered” in that it has been modified to comprise or express a nucleic acid sequence which is not naturally encoded by the cell.
  • Methods for engineering cells are known in the art and include but are not limited to genetic modification of cells e.g. by transduction such as retroviral or lentiviral transduction, transfection (such as transient transfection - DNA or RNA based) including lipofection, polyethylene glycol, calcium phosphate and electroporation. Any suitable method may be used to introduce a nucleic acid sequence into a cell.
  • nucleic acid sequence encoding secreted factor which is capable of binding a transforming growth factor beta receptor fl ⁇ R) and disrupting its interaction with transforming growth factor beta (TQRb) is not naturally expressed by a corresponding, unmodified cell.
  • Engineered cells according to the present invention may be generated by introducing DNA or RNA coding a CAR, TCR and/or antibody by one of many means including transduction with a viral vector, transfection with DNA or RNA.
  • Cells may be activated and/or expanded prior to the transfection or transduction, for example by treatment with an anti-CD3 monoclonal antibody or both anti-CD3 and anti-CD28 monoclonal antibodies.
  • activated means that a cell has been stimulated, causing the cell to proliferate, differentiate or initiate an effector function.
  • Methods for measuring cell activation include, for example, measuring the expression of activation markers by flow cytometry, such as the expression of CD69, CD25, CD38 or HLA-DR or measuring intracellular cytokines.
  • expansion means that a cell or population of cells has been induced to proliferate.
  • the expansion of a population of cells may be measured for example by counting the number of cells present in a population.
  • the phenotype of the cells may be determined by methods known in the art such as flow cytometry.
  • the cell may be an“immune effector cell” which is a cell of the immune system which responds to a stimulus and effects a change.
  • Immune effector cells include T cells (such as an alpha-beta T cell or a gamma-delta T cell), a B cells (such as a plasma cell), a Natural Killer (NK) cells or a macrophages.
  • Cytolytic immune cell as used herein is a cell which directly kills other cells. Cytolytic cells may kill cancerous cells; virally infected cells or other damaged cells. Cytolytic immune cells include T cells and Natural killer (NK) cells.
  • Cytolytic immune cells can be T cells or T lymphocytes which are a type of lymphocyte that play a central role in cell-mediated immunity.
  • T cells can be distinguished from other lymphocytes, such as B cells and NK cells, by the presence of a TCR on their cell surface.
  • Cytolytic T cells (TC cells, or CTLs) destroy virally infected cells and tumour cells, and are also implicated in transplant rejection.
  • CTLs express the CD8 at their surface.
  • CTLs may be known as CD8+ T cells. These cells recognize their targets by binding to antigen associated with MHC class I, which is present on the surface of all nucleated cells.
  • MHC class I which is present on the surface of all nucleated cells.
  • IL-10 adenosine and other molecules secreted by regulatory T cells, the CD8+ cells can be inactivated to an anergic state, which prevent autoimmune diseases such as experimental autoimmune encephalomyelitis.
  • the cell of the present invention may be a T-cell.
  • the T cell may be an alpha-beta T cell.
  • the T cell may be a gamma-delta T cell.
  • Natural Killer Cells are a type of cytolytic cell which form part of the innate immune system. NK cells provide rapid responses to innate signals from virally infected cells in an MHC independent manner.
  • NK cells (belonging to the group of innate lymphoid cells) are defined as large granular lymphocytes (LGL) and constitute the third kind of cells differentiated from the common lymphoid progenitor generating B and T lymphocytes. NK cells are known to differentiate and mature in the bone marrow, lymph node, spleen, tonsils and thymus where they then enter into the circulation.
  • LGL large granular lymphocytes
  • the cell of the present invention may be a wild-type killer (NK) cell.
  • the cell of the present invention may be a cytokine induced killer cell.
  • the cell may be derived from a patient’s own peripheral blood (1st party), or in the setting of a haematopoietic stem cell transplant from donor peripheral blood (2nd party), or peripheral blood from an unconnected donor (3rd party).
  • T or NK cells for example, may be activated and/or expanded prior to being transduced with nucleic acid molecule(s) encoding the polypeptides of the invention, for example by treatment with an anti-CD3 monoclonal antibody.
  • the cell may be derived from ex vivo differentiation of inducible progenitor cells or embryonic progenitor cells to T cells.
  • an immortalized T-cell line which retains its lytic function may be used.
  • the cell may be a haematopoietic stem cell (HSC).
  • HSCs can be obtained for transplant from the bone marrow of a suitably matched donor, by leukapheresis of peripheral blood after mobilization by administration of pharmacological doses of cytokines such as G-CSF [peripheral blood stem cells (PBSCs)], or from the umbilical cord blood (UCB) collected from the placenta after delivery.
  • cytokines such as G-CSF [peripheral blood stem cells (PBSCs)]
  • PBSCs peripheral blood stem cells
  • URB umbilical cord blood
  • the marrow, PBSCs, or UCB may be transplanted without processing, or the HSCs may be enriched by immune selection with a monoclonal antibody to the CD34 surface antigen.
  • the cell of the present invention may express a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • Classical CARs are chimeric type I trans-membrane proteins which connect an extracellular antigen-recognizing domain (binder) to an intracellular signalling domain (endodomain).
  • the binder is typically a single-chain variable fragment (scFv) derived from a monoclonal antibody (mAb), but it can be based on other formats which comprise an antibody-like antigen binding site or on a ligand for the target antigen.
  • mAb monoclonal antibody
  • a spacer domain may be necessary to isolate the binder from the membrane and to allow it a suitable orientation.
  • a common spacer domain used is the Fc of lgG1. More compact spacers can suffice e.g. the stalk from CD8a and even just the lgG1 hinge alone, depending on the antigen.
  • a trans membrane domain anchors the protein in the cell membrane and connects the spacer to the endodomain.
  • TNF receptor family endodomains such as the closely related 0X40 and 4-1 BB which transmit survival signals.
  • CARs have now been described which have endodomains capable of transmitting activation, proliferation and survival signals.
  • CAR-encoding nucleic acids may be transferred to T cells using, for example, retroviral vectors.
  • retroviral vectors In this way, a large number of antigen-specific T cells can be generated for adoptive cell transfer.
  • the CAR binds the target-antigen, this results in the transmission of an activating signal to the T-cell it is expressed on.
  • the CAR directs the specificity and cytotoxicity of the T cell towards cells expressing the targeted antigen.
  • the antigen-binding domain is the portion of a CAR which recognizes antigen.
  • the antigen-binding domain may comprise: a single-chain variable fragment (scFv) derived from a monoclonal antibody; a wild-type ligand of the target antigen; a peptide with sufficient affinity for the target; a single domain binder such as a camelid; an artificial binder single as a Darpin; or a single-chain derived from a T-cell receptor.
  • scFv single-chain variable fragment
  • CARs have also been described in which the antigen-binding domain is based on a ligand for the target antigen.
  • WO2015/052538 describes a BCMA-specific CAR in which the binding domain is based on a proliferation-inducing ligand (APRIL), rather than a BCMA-binding antibody.
  • APRIL proliferation-inducing ligand
  • the antigen-binding domain may bind to a tumour associated antigens (TAA).
  • TAA tumour associated antigens
  • Various TAAs have been described as being potentially suitable for targeting with a CAR or engineered TCR for cancer therapy, some of which are shown in the following Table 1.
  • the transmembrane domain is the sequence of a CAR that spans the membrane. It may comprise a hydrophobic alpha helix.
  • the transmembrane domain may be derived from CD28, which gives good receptor stability.
  • the CAR or transgenic TCR expressed by the cell of the present invention may comprise a signal peptide so that when it is expressed in a cell, such as a T-cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface, where it is expressed.
  • the core of the signal peptide may contain a long stretch of hydrophobic amino acids that has a tendency to form a single alpha-helix.
  • the signal peptide may begin with a short positively charged stretch of amino acids, which helps to enforce proper topology of the polypeptide during translocation.
  • At the end of the signal peptide there is typically a stretch of amino acids that is recognized and cleaved by signal peptidase.
  • Signal peptidase may cleave either during or after completion of translocation to generate a free signal peptide and a mature protein.
  • the free signal peptides are then digested by specific proteases.
  • the CAR may comprise a spacer sequence to connect the antigen-binding domain with the transmembrane domain.
  • a flexible spacer allows the antigen-binding domain to orient in different directions to facilitate binding.
  • the spacer sequence may, for example, comprise an lgG1 Fc region, an lgG1 hinge or a human CD8 stalk or the mouse CD8 stalk.
  • the spacer may alternatively comprise an alternative linker sequence which has similar length and/or domain spacing properties as an lgG1 Fc region, an lgG1 hinge or a CD8 stalk.
  • a human lgG1 spacer may be altered to remove Fc binding motifs.
  • the intracellular signalling domain is the signal-transmission portion of the CAR.
  • CD3-zeta endodomain which contains 3 ITAMs. This transmits an activation signal to the T cell after antigen is bound.
  • CD3-zeta may not provide a fully competent activation signal and additional co stimulatory signalling may be needed.
  • chimeric CD28 and 0X40 can be used with CD3-Zeta to transmit a proliferative / survival signal, or all three can be used together.
  • the intracellular signalling domain may be or comprise a T cell signalling domain.
  • the intracellular signalling domain may comprise one or more immunoreceptor tyrosine- based activation motifs (ITAMs).
  • ITAM immunoreceptor tyrosine- based activation motifs
  • An ITAM is a conserved sequence of four amino acids that is repeated twice in the cytoplasmic tails of certain cell surface proteins of the immune system.
  • the motif contains a tyrosine separated from a leucine or isoleucine by any two other amino acids, giving the signature YxxL/l. Two of these signatures are typically separated by between 6 and 8 amino acids in the tail of the molecule (Yxxl_/lx (6-8) Yxxl_/l).
  • ITAMs are important for signal transduction in immune cells. Hence, they are found in the tails of important cell signalling molecules such as the CD3 and z-chains of the T cell receptor complex, the CD79 alpha and beta chains of the B cell receptor complex, and certain Fc receptors.
  • the tyrosine residues within these motifs become phosphorylated following interaction of the receptor molecules with their ligands and form docking sites for other proteins involved in the signalling pathways of the cell.
  • the intracellular signalling domain component may comprise, consist essentially of, or consist of the O ⁇ 3-z endodomain, which contains three ITAMs.
  • the O ⁇ 3-z endodomain transmits an activation signal to the T cell after antigen is bound.
  • the intracellular signalling domain may comprise additional co-stimulatory signalling.
  • 4-1 BB also known as CD137
  • CD28 and 0X40 can be used with O ⁇ 3-z to transmit a proliferative / survival signal.
  • the endodomain may comprise: (i) an ITAM-containing endodomain, such as the endodomain from CD3 zeta; and/or
  • a co-stimulatory domain such as the endodomain from CD28 or ICOS;
  • a domain which transmits a survival signal for example a TNF receptor family endodomain such as OX-40, 4-1 BB, CD27 or GITR.
  • the CAR of the cell of the present invention may therefore comprise an antigen-binding component comprising an antigen binding domain and a transmembrane domain; which is capable of interacting with a separate intracellular signalling component comprising a signalling domain.
  • the vector of the invention may express a chimeric receptor signalling system comprising such an antigen-binding component and intracellular signalling component.
  • the cell of the invention may express a transgenic T-cell receptor (TCR).
  • TCR transgenic T-cell receptor
  • the TCR is a molecule found on the surface of T cells which is responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex (MHC) molecules.
  • MHC major histocompatibility complex
  • the TCR is a heterodimer composed of two different protein chains.
  • the TCR in 95% of T cells the TCR consists of an alpha (a) chain and a beta (b) chain (encoded by TRA and TRB, respectively), whereas in 5% of T cells the TCR consists of gamma and delta (g/d) chains (encoded by TRG and TRD, respectively).
  • the T lymphocyte When the TCR engages with antigenic peptide and MHC (peptide/MHC), the T lymphocyte is activated through signal transduction.
  • antigens recognized by the TCR can include the entire array of potential intracellular proteins, which are processed and delivered to the cell surface as a peptide/MHC complex.
  • heterologous TCR molecules it is possible to engineer cells to express heterologous (i.e. non-native) TCR molecules by artificially introducing the TRA and TRB genes; or TRG and TRD genes into the cell using a vector.
  • the genes for engineered TCRs may be reintroduced into autologous T cells and transferred back into patients for T cell adoptive therapies.
  • Such‘heterologous’ TCRs may also be referred to herein as‘transgenic TCRs’.
  • the transgenic TCR for use in the present invention may recognise a tumour associated antigen (TAA) when fragments of the antigen are complexed with major histocompatibility complex (MHC) molecules on the surface of another cell.
  • TAA tumour associated antigen
  • MHC major histocompatibility complex
  • the transgenic TCR for use in the present invention may recognise a TAA listed in Table 1.
  • the present invention provides a nucleic acid sequence which encodes a domain antibody (dAb) which binds transforming growth factor beta receptor type II (TbRII) and comprises one of the following sets of complementarity determining regions (CDRs):
  • dAb domain antibody
  • TbRII transforming growth factor beta receptor type II
  • the nucleic acid sequence may encode a dAb which comprises one of the sequences shown as SEQ ID No. 13, 14, 15 or 16.
  • the present invention provides a nucleic acid construct which comprises:
  • kits comprising nucleic acid sequences according to the present invention.
  • the kit may comprise
  • T R transforming growth factor beta receptor
  • polynucleotide As used herein, the terms“polynucleotide”,“nucleotide”, and“nucleic acid” are intended to be synonymous with each other.
  • the nucleic acid construct may comprise a plurality of nucleic acid sequences which encode a secreted factor or antibody which is capable of binding a transforming growth factor beta receptor O ⁇ R); and a CAR or transgenic TCR.
  • the nucleic acid construct may comprise two, three, four or more nucleic acid sequences which encode different components of the invention.
  • the plurality of nucleic acid sequences may be separated by co-expression sites as defined herein.
  • polynucleotides and nucleic acids can encode the same polypeptide as a result of the degeneracy of the genetic code.
  • skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides described herein to reflect the codon usage of any particular host organism in which the polypeptides are to be expressed.
  • the polynucleotides of the present invention are codon optimised to enable expression in a mammalian cell, in particular an immune effector cell as described herein.
  • Nucleic acids according to the invention may comprise DNA or RNA. They may be single- stranded or double-stranded. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the use as described herein, it is to be understood that the polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of interest.
  • variant in relation to a nucleotide sequence or amino acid sequence includes any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid(s) from or to the sequence.
  • a co-expression site is used herein to refer to a nucleic acid sequence enabling co expression of nucleic acid sequences encoding a secreted factor or antibody which is capable of binding a transforming growth factor beta receptor (T R) and a CAR or transgenic TCR according to the present invention.
  • T R transforming growth factor beta receptor
  • the same co-expression site may be used.
  • the co-expression site may be a cleavage site.
  • the cleavage site may be any sequence which enables the two polypeptides to become separated.
  • the cleavage site may be self cleaving, such that when the polypeptide is produced, it is immediately cleaved into individual peptides without the need for any external cleavage activity.
  • cleavage is used herein for convenience, but the cleavage site may cause the peptides to separate into individual entities by a mechanism other than classical cleavage.
  • FMDV Foot-and-Mouth disease virus
  • various models have been proposed for to account for the “cleavage” activity: proteolysis by a host-cell proteinase, autoproteolysis or a translational effect (Donnelly et al (2001) J. Gen. Virol. 82:1027-1041).
  • the exact mechanism of such “cleavage” is not important for the purposes of the present invention, as long as the cleavage site, when positioned between nucleic acid sequences which encode proteins, causes the proteins to be expressed as separate entities.
  • the cleavage site may be a furin cleavage site.
  • Furin is an enzyme which belongs to the subtilisin-like proprotein convertase family. The members of this family are proprotein convertases that process latent precursor proteins into their biologically active products.
  • Furin is a calcium-dependent serine endoprotease that can efficiently cleave precursor proteins at their paired basic amino acid processing sites. Examples of furin substrates include proparathyroid hormone, transforming growth factor beta 1 precursor, proalbumin, pro-beta-secretase, membrane type-1 matrix metalloproteinase, beta subunit of pro-nerve growth factor and von Willebrand factor.
  • Furin cleaves proteins just downstream of a basic amino acid target sequence (canonically, Arg-X-(Arg/Lys)-Arg') and is enriched in the Golgi apparatus.
  • the cleavage site may be a Tobacco Etch Virus (TEV) cleavage site.
  • TSV Tobacco Etch Virus
  • TEV protease is a highly sequence-specific cysteine protease which is chymotrypsin-like proteases. It is very specific for its target cleavage site and is therefore frequently used for the controlled cleavage of fusion proteins both in vitro and in vivo.
  • the consensus TEV cleavage site is ENLYFQ ⁇ S (where‘V denotes the cleaved peptide bond).
  • Mammalian cells such as human cells, do not express TEV protease.
  • the present nucleic acid construct comprises a TEV cleavage site and is expressed in a mammalian cell - exogenous TEV protease must also expressed in the mammalian cell.
  • the cleavage site may encode a self-cleaving peptide.
  • A‘self-cleaving peptide’ refers to a peptide which functions such that when the polypeptide comprising the proteins and the self cleaving peptide is produced, it is immediately“cleaved” or separated into distinct and discrete first and second polypeptides without the need for any external cleavage activity.
  • the self-cleaving peptide may be a 2A self-cleaving peptide from an aphtho- or a cardiovirus.
  • the primary 2A/2B cleavage of the aptho- and cardioviruses is mediated by 2A “cleaving” at its own C-terminus.
  • apthoviruses such as foot-and-mouth disease viruses (FMDV) and equine rhinitis A virus
  • the 2A region is a short section of about 18 amino acids, which, together with the N-terminal residue of protein 2B (a conserved proline residue) represents an autonomous element capable of mediating“cleavage” at its own C-terminus (Donelly et al (2001) as above).
  • 2A-like sequences have been found in picornaviruses other than aptho- or cardioviruses, ‘picornavirus-like’ insect viruses, type C rotaviruses and repeated sequences within Trypanosoma spp and a bacterial sequence (Donnelly et al., 2001) as above.
  • the co-expression sequence may be an internal ribosome entry sequence (IRES).
  • the co expressing sequence may be an internal promoter.
  • the present invention also provides a vector, or kit of vectors which comprises one or more nucleic acid sequence(s) or nucleic acid construct(s) of the invention.
  • a vector may be used to introduce the nucleic acid sequence(s) or construct(s) into a host cell so that it expresses a secreted factor or antibody and a CAR or transgenic TCR as defined herein.
  • the vector may comprise a plurality of nucleic acid sequences which encode different components as provided by the present invention.
  • the vector may comprise two, three, four or more nucleic acid sequences which encode different components, such as the secreted factor or antibody which binds TbR and a CAR or transgenic TCR.
  • the plurality of nucleic acid sequences may be separated by co expression sites as defined herein.
  • the vector may, for example, be a plasmid or a viral vector, such as a retroviral vector or a lentiviral vector, or a transposon based vector or synthetic mRNA.
  • the vector may be capable of transfecting or transducing a cell.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a cell according to the present invention or a cell obtainable (e.g. obtained) by a method according to the present invention.
  • the present invention also provides a pharmaceutical composition comprising a domain antibody (dAb), cell or plurality of cells as defined herein.
  • dAb domain antibody
  • the invention relates to a pharmaceutical composition containing a cell according to the present invention.
  • the pharmaceutical composition may additionally comprise a pharmaceutically acceptable carrier, diluent or excipient.
  • the pharmaceutical composition may optionally comprise one or more further pharmaceutically active polypeptides and/or compounds.
  • Such a formulation may, for example, be in a form suitable for intravenous infusion.
  • the present invention provides a method for treating and/or preventing a disease which comprises the step of administering a cell or plurality of cells or pharmaceutical composition according to the invention to a subject.
  • the present methods for treating and/or preventing a disease may comprise administering a cell according to the present invention (for example in a pharmaceutical composition as described above) to a subject.
  • the present invention also provides a method for treating and/or preventing a disease in a subject which subject comprises cells of the invention, which method comprises the step of administering an agent to the subject wherein the agent is capable of controlling the secretion or activity of secreted factor or antibody.
  • this method involves administering an agent to a subject which already comprises cells of the present invention.
  • the method for treating and/or preventing a disease may comprise the step of administering an agent which inhibits the secretion or activity of the secreted factor or antibody to a subject to which the cells according to the present invention have been administered.
  • the method for treating and/or preventing a disease may comprise the step of administering an agent which increases the secretion or activity of the secreted factor or antibody to a subject to which the cells according to the present invention have been administered.
  • a method for treating a disease relates to the therapeutic use of the cells of the present invention.
  • the cells may be administered to a subject having an existing disease or condition in order to lessen, reduce or improve at least one symptom associated with the disease and/or to slow down, reduce or block the progression of the disease.
  • the method for preventing a disease relates to the prophylactic use of the cells of the present invention.
  • the cells may be administered to a subject who has not yet contracted the disease and/or who is not showing any symptoms of the disease to prevent or impair the cause of the disease or to reduce or prevent development of at least one symptom associated with the disease.
  • the subject may have a predisposition for, or be thought to be at risk of developing, the disease.
  • the method may involve the steps of:
  • the nucleic acid construct, vector(s) or nucleic acids may be introduced by transduction or transfection.
  • the cell may be autologous or allogenic.
  • the methods provided by the present invention for treating a disease may involve monitoring the progression of the disease and/or any toxic activity.
  • the methods provided by the present invention for treating a disease may involve monitoring the progression of the disease and monitoring any toxic activity and adjusting the dose of the agent administered to the subject to provide acceptable levels of disease progression and toxic activity.
  • “Monitoring the progression of the disease” means to assess the symptoms associated with the disease over time to determine if they are reducing/improving or increasing/worsening.
  • Toxic activity relates to adverse effects caused by the cells of the invention following their administration to a subject. Toxic activities may include, for example, immunological toxicity, biliary toxicity and respiratory distress syndrome.
  • the dose of the agent administered to a subject, or the frequency of administration may be altered in order to provide an acceptable level of both disease progression and toxic activity.
  • the specific level of disease progression and toxic activities determined to be ‘acceptable’ will vary according to the specific circumstances and should be assessed on such a basis.
  • the agent may be administered in the form of a pharmaceutical composition.
  • the pharmaceutical composition may additionally comprise a pharmaceutically acceptable carrier, diluent or excipient.
  • the pharmaceutical composition may optionally comprise one or more further pharmaceutically active polypeptides and/or compounds.
  • Such a formulation may, for example, be in a form suitable for intravenous infusion.
  • the present invention provides a cell or pharmaceutical composition according to the present invention, a nucleic acid construct according to the present invention for use in treating and/or preventing a disease.
  • the present invention also relates to the use of a cell according to the present invention for the manufacture of a medicament for the treatment and/or prevention of a disease.
  • the disease to be treated and/or prevented by the method of the present invention may be cancer.
  • the cancer may be a cancer such as neuroblastoma, multiple myeloma, prostate cancer, bladder cancer, breast cancer, colon cancer, endometrial cancer, kidney cancer (renal cell), leukaemia, lung cancer, melanoma, non-Hodgkin lymphoma, pancreatic cancer, and thyroid cancer.
  • the cancer may be neuroblastoma.
  • the cancer may be multiple myeloma.
  • the cancer may be prostate cancer.
  • the cell of the present invention may be capable of killing target cells, such as cancer cells.
  • the target cell may be recognisable by expression of a TAA, for example the expression of a TAA provided above in Table 1.
  • the cancer may be a cancer listed in Table 1.
  • the administration of a cell or pharmaceutical composition according to the present invention can be accomplished using any of a variety of routes that make the active ingredient bioavailable.
  • the active ingredient can be administered by oral and parenteral routes, intranasally, intraperitoneally, intravenously, subcutaneously, transcutaneously or intramuscularly.
  • the cells of the present invention may be generated by introducing DNA or RNA coding for the secreted factor or antibody as defined herein, by one of many means including transduction with a viral vector, transfection with DNA or RNA.
  • the cell of the invention may be made by:
  • the cell may be transduced or transfected in vitro or ex vivo.
  • the cell may be from a sample isolated from a subject.
  • the present invention also provides a method of rendering a cell less susceptible to TQRb.
  • the method may comprise introducing to a cell (e.g. by transduction or transfection): the polynucleotide according to the present invention, the nucleic acid construct or vector according to the present invention, a first and second nucleic acid sequence as defined herein, a vector or a first and second vector as defined herein.
  • the method of rendering a cell less susceptible to TQRb signalling may comprise maintaining the cell under conditions which allow the expression of the secreted factor or antibody.
  • the present invention further relates to the use of secreted factor which binds TbE to render an immune effector cell less susceptible to TQRb.
  • a library of single domain antibodies was generated by immunising llamas with TQEbRII- Llama Fc, generating a phage display library, panning with TQRbRII-IiuGhqh Fc and eluting either with TQRb (competitive elution) or trypsin.
  • TQRbRII Binding to TQRbRII was investigated using a titration ELISA. Briefly, dAbs were expressed and His-purified and applied to plates which had previously been coated with antigen (TGFbRII-Fc) at 1 pg/ml. Plates coated with CD19-Fc were used as a control. Titrations were performed with various concentrations of dAb, starting at 10pg/ml and decreasing two fold (so 5, 2.5, 1.25 pg/ml etc).
  • the three related dAb binders (C6, E11 and G5) have similar affinities, all of which are in the sub-nanomolar range.
  • the fourth dAb (H3) has a very fast off rate.
  • T ⁇ Rb binding to T ⁇ RbRII can be competed by dAb blockade of TGFbRII, using a competition ELISA.
  • Plates were coated with T ⁇ RbRII-Ro (1 ug/ml) as described above and the dAbs were added at 0.25ug/ml.
  • the capacity of soluble TQRb to bind plate-bound TGFbRII was then tested by adding TQRb at 2.5ug/ml with 2 fold dilutions.
  • TQRb-B ⁇ o ⁇ h followed by Avadin HRP was used as a positive control and the results are shown in Figure 9. All four dAbs, including H3, competed with TQRb for binding to TGFbRII.
  • FIG. 10A is a schematic diagram illustrating the results which would be expected if soluble TGFb out-competed the dAb binder for binding TGFbRII.
  • Incremental response units (RU) of the sensograms are shown.
  • the first RU increase represents anti- TGFbRII VHH-Fc binding to the chip surface.
  • the second RU increase represents soluble TGFbRII binding to the captured antibody.
  • the third RU increase represents the binding of TGFbeta to the captured receptor.
  • Sensograms for the TGFbeta 0 and 300nM injections are superimposed (blue and yellow, respectively) to facilitate identification of TGFbeta 1 binding event.
  • the results for clone E11 are shown in Figure 10B.
  • the addition of T ⁇ Rb has no effect - there is no detectable third RU increase - indicating that E11-Fc outcompetes TGFb for binding to TGFbRII.
  • PBMCs were either left untransfected or transfected with a plasmid to co-express a) an RQR8 marker gene and b) a second generation GD2 CAR (HUK666-CD28z); or a) an RQR8 marker gene, b) a GD2 CAR and c) a dominant negative version of TGFbRII.
  • the plasmids used were as follows:
  • the cells were then incubated with each dAb-Fc conjugate and binding was analysed using flow cytometry and a murine Fc (APC).
  • APC murine Fc
  • T-cells were created co-expressing a CAR and a secreted anti-TGFbRII dAb.
  • T cells lines K562, Jurkat, SupT1 and 293 T-cells
  • a plasmid expressing the CAR and marker gene as described in Example 1
  • a plasmid expressing a dAb-Fc construct one of C6, G5, E11 and H3.
  • expression of dAb-Fc in the supernatant was analysed by ELISA using TGFbRII-hFc coated plates. Expression of dAb-Fc was detected using murine anti-Fc-HRP. All four cell lines were shown to successfully secrete all four dAbs (data not shown).
  • Example 3 The effect of anti-TGFBRII dAb secretion on CAR-mediated target cell killing T ⁇ Rb has immunosuppressive effects on T cells.
  • Blockade of TGFbRII aims to abrogate the effect of tumour secreted T ⁇ Rb by competitively inhibiting receptor binding and thus activation on T cells.
  • CAR-expressing T cells were used in cytotoxicity assays in the presence or absence of one of the four anti-TGFbRII dAbs.
  • the cytotoxicity assay was set up at a 1 :8 effectortarget (E:T) cell ratio using SupT1 expressing the antigen as target cells in presence or absence of recombinant human T ⁇ Rb1 (10 ng/ml final concentration) in 96-well plates.
  • Non-transduced T cells were used in co-cultures with targets as a negative control.
  • the anti TGFbRII dAb were produced in CHO cells and purified, and then added to the co-culture at 100 ng/ml.
  • CAR-mediated cytotoxicity was assessed by flow cytometry after 5 days.
  • T cells were identified from target cells by anti CD3 antibody, and a viability dye (7AAD) was used to separate live cells from dead cells using an iQue Screener PLUS flow cytometer. Counting beads were added to each sample as quality control. The number of live target cells remaining in the cultures were enumerated and normalized and the percentage cytotoxicity relative to control cultures calculated.
  • 7AAD viability dye
  • T ⁇ Rb1 inhibited target cell killing by the T-cells expressing the CAR alone, with more than 50% of target cells surviving at the 5 day point.
  • T cells expressing the same CAR which also secreted one of the four anti- TGFbRII dAbs was found to show resistance to such inhibition (Figure 12).
  • the transduced T cell population is labelled with the dye Cell Trace Violet (CTV), a fluorescent dye which is hydrolysed and retained within the cell. It is excited by the 405nm (violet) laser and fluorescence can be detected in the pacific blue channel.
  • CTV Cell Trace Violet
  • the T-cells are resuspended at 2x10 6 cells per ml in PBS, and 1 ul/ml of 5mM CTV is added.
  • the T-cells are incubated with the CTV for 20 minutes at 37°C. Subsequently, the cells are quenched by adding 5V of complete media. After a 5 minutes incubation, the T-cells are washed and resuspended in 2ml of complete media. An additional 10 minute incubation at room temperature allows the occurrence of acetate hydrolysis and retention of the dye.
  • Labelled T-cells are co-cultured with GD2 expressing target cells for four or seven days n order to investigate the function of the aTGFBeta dAb modules in the vector, target and effector cells are incubated in the presence or absence of 10ng/ml TGF Beta.
  • the assay is carried out in a 96-well plate in 0.2 ml total volume using 5x10 4 transduced T- cells per well and varying numbers of target cells (ratios - 1 :2 and 1 :8).
  • the T-cells are analysed by flow cytometry to measure the dilution of the CTV which occurs as the T-cells divide.
  • the number of T-cells present at the end of the co culture is calculated and expressed as a fold of proliferation compared to the input number of T cells.
  • Cytokine secretion is investigated following co-culture of the CAR-expressing T cells secreting each of the four anti-TGFbRII dAbs with target cells as described in Example 3.
  • IL2 and IFN y are assayed using ELISA assay kits according to the manufacturer’s instructions. Briefly, a double antibody sandwich detection ELISA is performed by coating anti-l L2 or ant-IFN y primary antibody onto a Nunc 96 well plate (1/1000 dilution and 50ul/well) or 1h. The plate is washed three times with 100ul of PBS 0.05% tween, blocked with 100ul PBS BSA (2%) for 1 h and washed again (100ul of PBS 0.05% tween).
  • the assay supernatant is added to the plate along with an appropriate titration standard and incubated for 1h before washing and detection using a secondary HRP conjugated anti-l L2 or IFN y antibody (1/1000 and 50ul/well). After a final wash the ELISA is developed with OPD and stopped with 1M NaOH (50ul each). The assays are read on a Varioskan lux plate reader at 450nm.
  • Anti-TGFbRII antibodies in soluble VHH format were expressed by transient expression on TG1 e.coli strain.
  • Supernatant from IPTG induced TG1 e.coli was purified using metal affinity chromatography.
  • a HisTrap 1 ml column was equilibrated with 5 column volumes of running buffer (300mM NaCI, 50 mM NaP04 pH 7.4).
  • Supernatant was applied to the column using AktaTM Pure system at a flow rate of 1 mL/min. Following application of supernatant, the column was washed with 20 column volumes of running buffer.
  • Anti-TGFbRII antibodies in murine lgG2a Fc format were expressed by transient expression on ExpiCHO cell lines by co-transfection of the relevant plasmid construct.
  • Supernatant from transfected CHO cells was purified using protein A affinity chromatography.
  • a HiTrap MabSelect SuRE 1 ml column was equilibrated with 5 column volumes of PBS pH 7.4.
  • Supernatant was applied to the column using AktaTM Pure system at a flow rate of 1 mL/min.
  • the column was washed with 20 column volumes of PBS.
  • Sample was then eluted from the column with 3 ml of IgG elution buffer (Pierce - 21004) at 1 mL/min and directly loaded onto 2 HiTrap 5 ml desalting columns, previously equilibrated in PBS, and collected on a 96-well plate using a fraction collector unit. Purity of antibody product was determined via SDS-PAGE.
  • Recombinant TGFbRII-Fc protein was immobilised on individual flow cells on a Series S CM5 sensor chip (GE Healthcare) previously functionalised with anti-human capture kit, to a density of 150-200 RU using a Biacore 8K instrument.
  • HBS-P + buffer was used as running buffer is all experimental conditions.
  • Recombinant purified VHH antibodies at known concentrations were used as the‘analyte’ and injected over the respective flow cells with 150 s contact time and 600s dissociation at 30 mI/minute of flow rate with a constant temperature of 25°C.
  • flow cell 1 was unmodified and used for reference subtraction.
  • a ⁇ concentration’ sensorgram of buffer alone was used as a double reference subtraction to factor for drift. Data were fit to a 1 :1 Langmuir binding model using local Rmax.

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Abstract

La présente invention concerne une cellule qui comprend un récepteur d'antigène chimérique (CAR) ou un récepteur de lymphocyte T transgénique (TCR) et sécrète un anticorps qui se lie à un récepteur de facteur de croissance transformant bêta (Tβ R).
PCT/GB2019/051284 2018-05-11 2019-05-10 Cellules car-t exprimant un anticorps à domaine unique anti-récepteur ii de tgf-bêta- inhibiteur WO2019215453A1 (fr)

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KR102314157B1 (ko) * 2020-01-10 2021-10-19 주식회사 뉴클릭스바이오 TGF-β 수용체에 특이적으로 결합하는 항체 및 이의 이용방법
WO2022172085A3 (fr) * 2021-02-15 2022-10-13 Takeda Pharmaceutical Company Limited Compositions de thérapie cellulaire et procédés pour moduler la signalisation du tgf-b

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